Phytopathology • 2017 • 107:804-815 • http://dx.doi.org/10.1094/PHYTO-02-17-0047-RVW

Rathayibacter toxicus,OtherRathayibacter Inducing Bacterial Head Blight of Grasses, and the Potential for Livestock Poisonings

Timothy D. Murray, Brenda K. Schroeder, William L. Schneider, Douglas G. Luster, Aaron Sechler, Elizabeth E. Rogers, and Sergei A. Subbotin

First author: Department of Pathology, Washington State University, Pullman, WA 99164; second author: Entomology, Plant Pathology and Nematology, University of Idaho, Moscow, ID 83844; third, fourth, fifth, and sixth authors: U.S. Department of Agriculture, Agricultural Research Service, Foreign Disease-Weed Science Research Unit, Ft. Detrick, MD 21702; and seventh author: Department of Food and Agriculture, 3294, Meadowview Road, Sacramento, CA 95832-1448. Accepted for publication 8 April 2017.

ABSTRACT

Rathayibacter toxicus, a Select Agent in the United States, is one of six recognized species in the genus Rathayibacter and the best known due to its association with annual ryegrass toxicity, which occurs only in parts of . The Rathayibacter species are unusual among phytopathogenic in that they are transmitted by anguinid seed gall and produce extracellular polysaccharides in infected resulting in bacteriosis diseases with common names such as yellow slime and bacterial head blight. R. toxicus is distinguished from the other species by producing corynetoxins in infected plants; toxin production is associated with infection by a bacteriophage. These toxins cause grazing feeding on infected plants to develop convulsions and abnormal gate, which is referred to as “staggers,” and often results in death of affected animals. R. toxicus is the only recognized Rathayibacter species to produce toxin, although reports of livestock deaths in the United States suggest a closely related toxigenic species may be present. A closely related but undescribed species, Rathayibacter sp. EV, originally isolated from Ehrharta villosa var. villosa in South Africa, is suspected of producing toxin. Many of the diseases caused by Rathayibacter species occur in arid areas and the extracellular polysaccharide they produce is believed to aid in their survival between crops. For example, R. “agropyri” was isolated from infected plant material after being stored for 50 years in a herbarium. Similarly, the anguinid vectors associated with these bacteria form seed galls in infected plants and are capable of surviving for very long periods of time under dry conditions. The addition of R. toxicus to the list of Select Agents has raised concern over its potential introduction and a realization that current diagnostic methods are inadequate to distinguish among Rathayibacter species. In addition, little is known about the Rathayibacter species and their seed gall vectors present in the United States.

Additional keywords: bacteriology, ecology and epidemiology, etiology, nematology.

Rathayibacter toxicus (Riley and Ophel 1992) Sasaki et al. 1998 are quarantine pests, to nearby host plants where it causes a was listed as a Plant Pathogen Select Agent under 7 CFR 331 by the bacteriosis disease of leaves and floral structures of several differ- U.S. Department of Agriculture (USDA) and Plant Health ent grass species, but primarily annual ryegrass ( rigidum Inspection Service in 2008 (Murray et al. 2015). Relatively few Gaud.). During colonization, R. toxicus produces corynetoxins that plant pathologists would have known of this bacterial plant can result in fatal poisoning of animals that graze on diseased plants, pathogen prior to its listing as a Select Agent, and fewer still would a syndrome now referred to as annual ryegrass toxicity (ARGT). To know that there are five other described species of Rathayibacter further complicate the relationship, Bird et al. (1980) and Stynes that cause similar diseases and have similar life cycles in other grass and Bird (1983) postulated the involvement of a bacteriophage in hosts. What is it about this Gram-positive bacterium that makes it toxin production. In Australia, this cross-domain pathogen has such a threat to be considered a Select Agent? R. toxicus is caused plant disease on over 10 million hectares with losses due to transmitted by seed gall nematodes ( sp.), some of which ARGT in 2010 estimated at $37 million USD (Carslake 2006; Kessell 2010). The potential for damage to the U.S. livestock industry resulted in listing of R. toxicus as a Select Agent in 2008 Corresponding author: T. Murray; E-mail address: [email protected] and relisting in 2012. The National Plant Disease Recovery System (NPDRS) subsequently developed a recovery plan for this pathogen Rathayibacter This article is in the public domain and not copyrightable. It may be freely (Murray et al. 2015). None of the other five described reprinted with customary crediting of the source. The American Phytopathological species have been shown to produce toxins in planta, although Society, 2017. reports exist of livestock poisonings in the United States and

804 PHYTOPATHOLOGY elsewhere after animals grazed on pastures where seed gall nema- for sheep feeding trials. Postmortem examinations also were todes were present (Cunningham and Hartley 1959; Galey et al. conducted on animals that died of staggers from farms and feeding 1997; Galloway 1961; Haag 1945; Jensen 1961; Kurochkina and trials. They found that all pastures contained Wimmera ryegrass Chizhov 1980; Shaw and Muth 1949). (L. rigidum) and had generally high nematode infestations. One This review will examine the literature relevant to the history and such pasture with 21% infected seed had the greatest sheep biology of Rathayibacter broadly because they represent a fasci- mortality. Experimentally, sheep that grazed on one of the infested nating group of cross-domain pathogens that frequently occur in pastures developed symptoms typical of staggers and died, as arid areas (Fig. 1A). As such, both the pathogens and their vectors did guinea pigs fed bacteria-infected seed. All had postmortem have developed specialized mechanisms for anhydrobiotic survival, symptoms including uncoordinated movement and tetanic spasms namely production of extracellular polysaccharides (EPS) (Fig. 1B that were consistent with those of animals that died of staggers and C) and the formation of galls (Fig. 2A and B), in many of these described previously. McIntosh et al. (1967) could not conclusively diseases. Readers primarily interested in R. toxicus are directed demonstrate whether the toxicity was due to the nematode or the to the reviews by McKay and Ophel (1993), Riley et al. (2014), and bacterium, but concluded that both were associated with ryegrass the NPDRS Recovery Plan (Murray et al. 2015), which discuss staggers. Lanigan et al. (1976) followed-up this research with the relevant literature and provide recommendations for control and animal feeding studies and concluded that the bacterial galls and not surveillance. the nematode galls were the source of toxicity. Gwynn and Hadlow (1971) expanded the reports of toxicity to ANNUAL RYEGRASS TOXICITY sheep grazing on Wimmera ryegrass to include pastures in Western Australia. They noted that the disease had occurred in four of the In 1956, Fisher (1977) received samples of annual ryegrass six previous years in one particular pasture. Berry and Wise (1975) (L. rigidum) from veterinary officers with the South Australia coined the term Wimmera rye grass toxicity, or WRGT, for the Department of Agriculture that were taken from a pasture where syndrome in animals associated with grazing pastures infested with sheep had died after feeding on the grass. Fisher found seed gall seed gall nematodes and bacteria and noted that there were 58 nematodes present in the sample and, while visiting the field 2 years outbreaks on 26 farms with over 3,300 dead sheep and 46 cattle. Up later, observed symptoms of bacterial head blight, which he to 60% of the ryegrass sampled from the 26 farms was infected with attributed to a Corynebacterium species. Nematode seed galls both Anguina sp. and Corynebacterium sp. (now Rathayibacter), picked from the sample and fed to laboratory mice resulted in the neither of which were identified to species. death of one with symptoms similar to those of the dead sheep. By Bryden et al. (1991) described the occurrence of a syndrome 1967, increases in the numbers of sheep dying annually and affected known as flood plain staggers (FPS) in cattle and sheep in New properties in South Australia were great enough that funds were South Wales, Australia. Samples of blown grass ( avenacea obtained to begin new research on the problem at the Waite C.C. Gmelin) were collected and used in feeding studies that Agricultural Institute (Fisher 1977). resulted in animals developing symptoms similar to ARGT. Like McIntosh et al. (1967) were the first to conclude that a toxin was ARGT, a nematode and Clavibacter sp. (now Rathayibacter) were present where sheep were dying after feeding on infested pastures. found in seed heads, and a corynetoxin was extracted from the They surveyed nine farms where staggers occurred and sampled bacterial galls (Cockrum and Edgar 1985). Davis et al. (1995) later grass from four of them. Samples were identified to plant species, reported that 1,722 cattle, 2,466 sheep, and 11 horses on 31 different evaluated for presence of (Steinbuch 1799) properties died as a result of FPS through April 1991. During the Filipjev 1936, separated into seed and stalk fractions for guinea pig same year, Finnie (1991) described a corynetoxin poisoning of feeding trials, and bulk samples were collected from one property sheep in South Australia that was associated with annual beard grass

FIGURE 1 Typical habitat and symptoms of bacterial head blight of grass (Agropyron sp.) collected in Idaho and Montana, and colony characteristics of the pathogen. A, Overview of roadside collection site with box showing the collection area, B, infected wheat grass head, C, close-up showing droplets of dried ooze (extracellular polysaccharide) on infected wheat grass head, and D, yellow-pigmented colonies of Rathayibacter “agropyri” growing on nutrient broth yeast extract agar.

Vol. 107, No. 7, 2017 805 (Polypogon monspeliensis (L.) Desf.) and referred to it as Stewart’s diagnosis was based on symptomatology of affected animals and the range syndrome (SRS) (McKay et al. 1993). According to Finnie presence of bacterial galls in hay fed to the horses. (1991), the syndrome was indistinguishable from ARGT and had Nogawa et al. (1997) reported an outbreak of annual ryegrass been known for about 20 years; Anguina sp. nematodes and intoxication in cattle and sheep in Japan. In this case, animals from corynetoxins were confirmed present in the sample from South farms were fed oat hay contaminated with annual ryegrass imported Australia. McKay et al. (1993) conclusively demonstrated that from Australia. ARGT was confirmed after detection of corynetox- Clavibacter toxicus was present in both Agrostis avenacea and ins in seed from the hay and feeding studies where cattle developed P.monspeliensis and thus responsible for both FPS and SRS; however, symptoms consistent with the syndrome. they also demonstrated that the nematode present in these grasses was a new species and not the same one present in annual ryegrass. OTHER ANIMAL POISONINGS Schneider (1981) noted the occurrence of ARGT in South Africa in two different districts. In one, Caledon, sheep grazed on a pasture Several reports have appeared describing poisonings of grazing composed of oats, ryegrass and vetch or fed hay harvested from the animals outside Australia with similarities to ARGT, but the pasture developed symptoms consistent with ARGTand died. In the presence of a toxigenic bacterium has not been confirmed in any of other, Bredasdorp, cattle deaths occurred on three different farms; those cases. In 1941, Mullins (1941) described a disease of sheep, in all cases, cattle had grazed on mixed pastures containing Italian cattle, and horses in New Zealand known as ryegrass staggers (L. multiflorum Lam.) and/or annual ryegrass, among other plant because it was found mostly in animals that had grazed on pastures species. Examination of hay or plant samples from each of the four containing predominately ryegrass. Cunningham and Hartley pastures revealed the presence of seed gall nematodes identified (1959) documented three cases of staggers that occurred in 1957. as Anguina agrostis, and a pale yellow, Gram-positive bacterium They were unable to determine the cause of the disease, which identified as Corynebacterium sp. was isolated. Feeding trials with occurred during autumn months, but believed it was associated with hay collected from some of the pastures and separated by plant a toxin of unknown origin in the perennial ryegrass (Lolium perenne species confirmed the toxicity was associated with the ryegrass and L.). No subsequent reports of annual ryegrass toxicity-like incidents not the other species present. are found from New Zealand. Grewar et al. (2009) described an outbreak of ARGT in horses in Several reports of animal poisonings in the United States with the Western Cape Province of South Africa, near Ceres and within similarities to ARGT have appeared. Haag (1945) reported mortality 10 km of one of the cattle outbreaks previously reported by of sheep in Oregon that had consumed screenings of Chewings fescue Schneider (1981). In this case, horses were fed oat hay contaminated (Festuca rubra subsp. commutata Gaud.). He obtained and fed with annual ryegrass; morbidity was limited to two pastures. The screenings from the same lot to rats and showed that the material

FIGURE 2 A, Yellowish bacterial galls and B, dark nematode galls in an inflorescence of Sporobolus cryptandrus (Torr.) A. Gray with bacterial head blight collected in Idaho.

806 PHYTOPATHOLOGY was highly toxic. Symptoms included paralysis of rear quarters grasses including ryegrass. There was no mention of examining the with swelling and discoloration of rear legs and extensive tissue grasses for seed gall nematodes or bacterial head blight. hemorrhages. Haag (1945) tested several other lots of screenings, Riley et al. (2004a) examined bacterial galls from old Chewings confirmed their toxicity to rats, and concluded there was a strong fescue samples produced in New Zealand and New Jersey and causal relationship between toxicity and Anguina agrostis seed gall concluded that the most likely cause of the livestock symptoms was nematode infestation. the presence of corynetoxin-like toxins produced by a variant of Shaw and Muth (1949) described several cases of cattle and sheep R. toxicus. This conclusion was based on (i) detection of R. toxicus poisonings in Oregon after feeding on various plants including in galls from New Zealand, but not the United States, using a perennial rye (L. perenne), pea vine silage, and Chewings fescue monoclonal antibody specific for R. toxicus; (ii) detection of screenings. Animals fed on the latter exhibited nervous system corynetoxin-like chemicals by enzyme-linked immunosorbent assay symptoms including falling, trembling, and incoordination. Feeding (ELISA) and high-performance liquid chromatography (HPLC) in studies of sheep with the fescue screenings resulted in symptomatology galls from both New Zealand and the United States, and in vitro and mortality similar to animals that fed on infested pastures. Anguina inhibition of an enzyme specifically inhibited by corynetoxins and agrostis was present in up to 20% of the seed screenings. The cause of related tunicaminyluracil antibiotics by extracts of each sample (Jago mortality was not confirmed, but presence of the nematode had the et al. 1983); and (iii) the toxin mixture in both samples was very similar, strongest association. but differed from that typical of R. toxicus galls (Anderton et al. 2004). Jensen (1961) reported the occurrence and distribution of the grass seed nematode (Anguina agrostis) infecting several bentgrass THE RATHAYIBACTER SPECIES species, Chewings fescue, and orchard grass in Oregon. He also mentioned the occurrence of livestock poisonings in animals fed Investigations of bacterial head blight of grasses, their causal Chewings fescue screenings containing nematode galls, and attributed agents, and the vectors involved with them, now known as it to the combined relationship between the nematode, a bacterium, Rathayibacter spp. and Anguina or Afrina spp. (B. L. Barrantes- and the host plant. However, there was no mention of isolation or Infante, B. K. Schroeder, S. A. Subbotin, and T. D. Murray, characterization of the bacterium. unpublished data), respectively, began in 1897 when Emerich Galloway (1961) described three cases of livestock poisoning Rathay´ described bacteriosis disease of orchard grass (Dactylis associated with feeding Chewings fescue seed screenings from a glomerata L.) in Germany and associated a bacterium with it single farm in Oregon. Toxicity was associated with the presence of (Rathay´ 1899; Smith 1914). Rathayibacter species historically heavy infestation of Anguina sp. seed gall nematode. Two types of were placed in Corynebacterium (Collins and Bradbury 1986), and galls were noted in the hay; a brownish one that contained nematode later in Clavibacter (Davis et al. 1984). The genus Corynebacterium larvae and another that contained a “yellow cheesy material” from originally contained Gram-positive, bacilliform-shaped bacteria which a Gram-positive bacterium resembling Corynebacterium sp. (Fig. 3A) with 2, 4-diaminobutyric acid (DAB) in their cell walls was isolated. Feeding studies conducted with chickens and rats (Carlson and Vidaver 1982; Collins and Bradbury 1986; Zgurskaya using hay extract and pellets made from the nematode-infested hay et al. 1993); however, since this was a heterogeneous genus demonstrated that toxicity was associated with an alcohol-soluble (Carlson and Vidaver 1982; Dye and Kemp 1977; Starr et al. 1975), substance (Galloway 1961). Kurochkina and Chizhov (1980) in Davis et al. (1984) proposed establishing the genus Clavibacter Russia reported animal poisoning after feeding on seed galls of for the Gram-positive, DAB-containing plant-pathogenic bacteria Anguina agrostis from Agrostis sp. and suggested an association (Zgurskaya et al. 1993). Plant pathogenicity was considered a with R. toxicus or a related bacterium producing similar toxins. distinguishing characteristic of the bacteria. Based on morphological Galey et al. (1997) described the occurrence of “staggers” in and physiological characteristics including menaquinone composi- cattle in the central valley of California. Incidence ranged from 5 to tion, along with phylogenetic analyses resulted in placement of some 50% of animals, and the clinical course and syndrome resembled coryneform species in separate genera (Stackebrandt et al. 1988). flood plain staggers. Investigation of three ranches where the losses Zgurskaya et al. (1993) subsequently proposed moving species that occurred revealed the pastures did not contain perennial grasses contain B2g peptidoglycan with DAB in the cell wall (Sasaki et al. usually associated with staggers, but they did contain other annual 1998), have MK-10 as the major menaquinone, phosphotidylglycerol

FIGURE 3 Scanning microscopic photo of A, Rathayibacter “agropyri” and B, infective juvenile of a seed gall nematode covered by Rathayibacter sp. (CA32). A and B, Scale bars 5 1 and 20 µm.

Vol. 107, No. 7, 2017 807 and diphosphotidylglycerol as the major phospholipids, saturated rathayi´ in honor of Rathay’s´ pioneering work (Smith 1914); it is the anteiso-15:0, anteiso-17:0, and iso-16:0 as the major branched fatty type species for Rathayibacter (Burkholder 1948). This disease, acids, and rhamnose and mannose as the major cell wall sugars to the commonly referred to as Rathay’s disease, was frequently found genus Rathayibacter. throughout the European continent and England and is associated The genus Rathayibacter currently contains six valid species: with seed as a source of inoculum (Dowson and d’Oliveira 1935). R. rathayi (Smith 1913) Zgurskaya et al. 1993; R. tritici (Carlson and Rathay’s disease is present and increasing in frequency in Oregon Vidaver 1982) Zgurskaya et al. 1993; R. iranicus (Carlson and Vidaver (Alderman et al. 2003, 2005). 1982) Zgurskaya et al. 1993; R. toxicus; R. caricis Dorofeeva et al. Rathayibacter “agropyri”. Around the same time as Rathay’s´ 2002; and R. festucae Dorofeeva et al. 2002. Three other species work, O’Gara (1915) described a bacterial disease of western have been identified but not validly described: R. “agropyri”(B.K. wheatgrass (Agropyron smithii Rydb.) in the Salt Lake Valley, Utah Schroeder, W. L. Schneider, D. G. Luster, A. Sechler, and T. D. with similar characteristics to those described on orchard grass Murray, unpublished data), Rathayibacter sp. EV (Riley et al. 2004b), by Rathay´ (1899). He subsequently described the bacterium as and Rathayibacter sp. TV (Vasilenko et al. 2016). All Rathayibacter Aplanobacter agropyri, suggesting yellow gum disease as the species identified and described to date arevectored by plant parasitic common name. O’Gara (1916) noted the absence of symptoms nematodes, a feature unusual among phytopathogenic bacteria. on other grass species in mixed stands and speculated that the R. toxicus occupies a basal position in the genus phylogeny and it bacterium may be spread by insects. In 1980, with no known type is most distinct from the other species based on the 16S rRNA gene strain in existence and the inability to distinguish it from R. rathayi sequence (Fig. 4) (Dorofeeva et al. 2002; Wellington 2009). Based (syn. Corynebacterium rathayi), R. “agropyri” (syn. Corynebacte- on preliminary data, we expect additional Rathayibacter species rium agropyri) was not included on the Approved List of Bacterial to be described in the near future (B. K. Schroeder, W. L. Schneider, Names (Skerman et al. 1980). Cultures resembling R. “agropyri” D. G. Luster, A. Sechler, and T. D. Murray, unpublished data) were isolated in 1982 from grass samples collected in the 1940s and (Starodumova et al. 2014) (Fig. 3). 1950s and from a sample of wheatgrass with yellow EPS (Fig. 1D) Rathayibacter rathayi. Rathay´ (1899) initially discovered collected in Montana in 1986 (Murray 1986). These cultures, cocksfoot grass, also known as orchard grass, exhibiting gummosis which were stored at the Washington State University Mycological near Vienna in 1897, but never observed similar symptoms on other Herbarium, Pullman, WA, were recently characterized physiolog- grass species growing in the vicinity of the infected plants. He ically and molecularly. The name R. “agropyri” has been proposed described affected plants as being somewhat stunted and covered to reestablish it as a valid species (B. K. Schroeder, W. L. Schneider, with a thick, lemon-yellow bacterial slime on the uppermost stems D. G. Luster, A. Sechler, and T. D. Murray, unpublished data). and leaves, and inflorescences (Smith 1914), a symptom associated . Hutchinson (1917) described a bacterial with many of the diseases caused by bacteria in this genus. Many disease (hereafter referred to as head blight) of wheat in the Punjab organisms were isolated from the slime including fungi and a of India that resembled the diseases described by Rathay´ and bacterium with lemon-yellow colonies. Although Rathay´ inoculated O’Gara, and which was known locally as tannan or tandu (also seedlings and mature orchard grass plants without producing known as tundu). The disease was known prior to 1908, but was not infection, he was convinced that the bacterium caused the disease. of major concern because it affected a small percentage of plants Smith (1913) later described the causal bacterium as Aplanobacter within fields. Based on comments by D. Milne, economic botanist

FIGURE 4 Phylogenetic relationships within phytopatho- genic Rathayibacter species (isolate, GenBank accession number): Bayesian 50% majority rule consensus tree from two runs as inferred from the analysis of the 16S rRNA gene sequence alignment under the GTR + I + G model. Posterior probabilities equal to, or more than, 60% are given for appropriate clades. Strains used in the analysis include the following: R. “agropyri” strain CA-4, R. caricis, R. festucae, R. iranicus, R. rathayi, Rathayibacter sp. TV strain VKM Ac-2596, R. toxicus (D84127), R. tritici, Rathayibacter sp. Leaf296, Rathayibacter sp. EV strain FH238, Rathayibacter sp. strain CA36, and Rathayibacter sp. strain CA32. The outgroups were Agromyces brachium, Agromyces rhizospherae, and Agro- myces ramosus.

808 PHYTOPATHOLOGY in the Punjab at that time, Hutchinson noted the possible association (1993) later erected the genus Rathayibacter and transferred of head blight with “eelworm” (nematode) damage, since it only Corynebacterium iranicus, Corynebacterium rathayi, Corynebacte- occurred in areas of the field where they occurred. Hutchinson rium tritici, and six strains isolated from annual grasses in Australia (1917) described the bacterium as Bacterium tritici (syn. Pseudo- that resembled Clavibacter toxicus to it. Sasaki et al. (1998) monas tritici), but was unable to successfully infect plants with the subsequently proposed R. toxicus comb.nov.onthebasisofcell bacterium. Milne (1919) later described earcockle disease of wheat wall peptidoglycan content. caused by the nematode Tylenchus scandens (5T. tritici;now Rathayibacter caricis. R. caricis was isolated from the phyllo- (Steinbuch 1799) Chitwood 1935). He noted that sphere of Carex sp. growing in the Central Chernozem Nature Park, some plants infected with the nematode had distorted heads Belgorod region, Russia (Dorofeeva et al. 2002). Although R. caricis and produced a slimy yellowish substance, but did not mention a was not found to be associated with any nematodes in this habitat, their bacterium associated with it. association cannot be excluded. A potential vector for this bacterium Fahmy and Mikhail (1925) described the occurrence and dis- could be Heteroanguina caricis (Solovyova and Krall 1983) Chizhov tribution of head blight of wheat caused by Pseudomonas tritici in and Subbotin 1985, which induces leaf galls on several Carex species Egypt. It was first reported to them in 1919, but was likely present and is widely distributed in Estonia, Latvia, and Lithuania, as well as before and believed to be introduced to Egypt on seed from India. several regions of Northwest Russia. Recently, Starodumova et al. Based on a survey in 1923, Fahmy and Mikhail (1925) observed that (2014) reported R. caricis from leaf galls induced by Mesoanguina affected fields often were in close proximity to one another and that picridis (Kirjanova 1944) Chizhov & Subbotin 1985 in Acroptilon damage within fields was clustered in patches. They also observed repens (L.) DC. from Uzbekistan. that the disease did not spread aerially between fields since healthy Rathayibacter festucae. R. festucae was isolated and described and diseased fields were sometimes adjacent to one another, which by Dorofeeva et al. (2002) from small leaf galls on Festuca rubra led them to consider seed as the source of infection. Fahmy and L. infected by the nematode Anguina graminis (Hardy 1850) Filipjev Mikhail (1925) described the early symptoms of the disease as 1936, collected in the Moscow region of Russia. This nematode wrinkled or twisted leaves appearing on lower leaves on the wheat parasitizes several Festuca species in Europe and northwestern plant with a bright yellow gummy substance now known to be EPS European part of Russia. exuding from these impacted plant structures. As the EPS dried, it Rathayibacter sp. EV. Rathayibacter sp. EV was reported in became a solid, almost clear droplet on the plant surface (Fig. 1C). 2004 by Riley et al. (2004b), who observed gummosis and leaf galls Fahmy and Mikhail (1925) further described the association of head caused by Anguina woodi Swart et al. 2004 on Ehrhata villosa blight with nematode damage (Tylenchus tritici) and demonstrated Schult. f. var. villosa in South Africa. The bacterium isolated from experimentally that the nematode was required to carry the bacte- these plants was identified as a species of Rathayibacter based on rium into the plant for infection, although experimental conditions physiological characteristics. However, DNA analysis of the and data were not presented. bacteria demonstrated only 70% homology with genomic DNA Carne (1926) described the occurrence of earcockle and head from other Rathayibacter species, suggesting the isolate was a new blight of wheat caused by Pseudomonas tritici in Western Australia. species. The bacterium adheres to juveniles of Anguina woodi, the He noted that earcockle was first reported in 1898, but that head leaf gall nematode of E. villosa var. villosa, but not to Anguina blight was not previously known in Western Australia. He also funesta or Anguina tritici (Riley et al. 2004b). Interestingly, in observed that earcockle did not form on plants with head blight, planta production of toxin was detected in a bioassay and this work suggesting that nematodes were the agent of their own destruction. suggests that the bacterium is a unique and previously undescribed Based on field experiments, Carne (1926) concluded that the species currently referred to as Rathayibacter sp. EV (Riley et al. nematodes do not travel more than a few inches from the galls in soil 2004b). and that the bacteria require the nematode to be carried into the plant Rathayibacter sp. TV. Rathayibacter sp. TV was isolated from and cause disease. leaves of Tanacetum vulgare L. infected with nematodes in the Main Rathayibacter iranicus. R. iranicus was originally isolated from Botanical Garden, Moscow, Russia, and a draft genome sequence of diseased wheat heads in Iran exhibiting symptoms characteristic this undescribed species was released in 2016 (Vasilenko et al. of Rathayibacter infection of grass heads (Scharif 1961). The 2016). Although it had 99.6% 16S rRNA gene sequence similarity bacterium was isolated from the honey-yellow slime from aborted with R. rathayi, R. iranicus, and R. tritici, the results from matrix- ovaries within infected wheat grains, but had discrepancies in assisted laser desorption ionization–time of flight mass spectra physiological characteristics compared with R. rathayi (Coryne- clustering and multilocus phylogenetic analysis (gyrB, recA, rpoB, bacterium rathayi) (Burkholder 1948, Dowson 1942; Sabet 1954), and ppk) suggested that it was a novel species (Vasilenko et al. R. “agropyri”(Corynebacterium agropyri) (O’Gara 1915, 1916), 2016). This was the first report of a Rathayibacter species associated and R. tritici (Burkholder 1948); consequently, it was described with the foliar nematode Aphelenchoides fragariae (Ritzema Bos as a new species, Corynebacterium iranicum (Scharif 1961), now 1891) Christie 1932, which belongs to the nematode order Aphe- R. iranicus (Zgurskaya et al. 1993). This pathogen appears to be lenchida, family Aphelenchoididae, whereas all other previously extremely limited in geographic distribution, having only been known nematode vectors of Rathayibacter species belong to the order identified on wheat in Iran in 1961 and more recently in Turkey in , family . This is significant because Aphelen- 2009 (Postnikova et al. 2009). choides fragariae is an important parasite of several agricultural crops Rathayibacter toxicus. Bird and Stynes (1977) concluded and numerous ornamentals with worldwide distribution. that the bacterial pathogen associated with ARGT belonged to Even though it is uncommon for a phytopathogenic bacterium to the Corynebacterium rathayi, Corynebacterium agropyri, and be vectored by a nematode, it should be noted that Rathayibacter Corynebacterium tritici group based on colony color and species are not the only bacteria inhabiting nematode galls symptomatology on the host plant, but there wasn’t enough data (Evtushenko et al. 1994). Several other members of the family for a full comparison and species designation. Although pre- have been characterized and described: Leifso- sumptively referred to as Corynebacterium rathayi in many reports nia poae (Evtushenko et al. 2000) from root galls induced by the (Riley 1987), Riley and Ophel (1992) described the bacterium as grass root gall nematode (Greeff 1872) Clavibacter toxicus sp. nov. based on serological properties, Paramanov 1968 on Poa annua L. (Evtushenko et al. 2000); Agreia allozyme electrophoretic mobility, bacteriophage sensitivity, adhe- bicolorata Evtushenko et al. 2001 and Agreia spp. from leaf galls sion to seed gall nematodes, biochemical tests, DNA base induced by Heteroanguina graminophila Evtushenko et al. 2001 on composition, and growth factor requirements. Zgurskaya et al. narrow reed grass, Calamagrostis neglecta (Ehrh.) Gaertn., B. Mey.

Vol. 107, No. 7, 2017 809 & Scherb. (Evtushenko et al. 2001; Starodumova et al. 2015); and and observations suggest that the bacteria are not able to invade three undescribed species of Plantibacter isolated from plant galls plants in the absence of nematodes, but advantages of the bacteria induced by Anguina agrostis, Aplanobacter agropyri Kirjanova to the nematodes are not evident; in fact, they may be negative. 1955, and Mesoanguina picridis (Evtushenko and Takeuchi 2006). For example, juvenile nematodes with attached bacteria moved significantly slower than nematodes without attached bacteria and NEMATODE VECTORS thus, have a lower chance of successfully invading a plant (Bird and Riddle 1984). The gall-forming nematodes of the subfamily Anguininae are Anguina funesta and Anguina paludicola are the two known obligate, highly specialized plant parasites that induce galls in natural nematode vectors of R. toxicus. The seed gall nematode various plant organs. Over 40 nominal species of gall-forming Anguina funesta was first described in 1973 as Anguina lolli in the nematodes have been described. Three species, Anguina tritici, Ph.D. thesis by Price, and later named Anguina funesta when it Anguina agrostis, and Anguina funesta, induce seed galls on some was formally described (Price et al. 1979b). Anguina funesta was cereals and grasses, and are considered of economic importance as considered a synonym of Anguina agrostis by several authors; agricultural and quarantine pests in some countries (Chizhov and however, Powers et al. (2001) and Subbotin et al. (2004) examined Subbotin 1990; Krall 1991). Several Anguina species are currently relationships within Anguina using internal transcribed spacer recognized as vectors of Rathayibacter species. Rathayibacter (ITS) rRNA gene sequences and concluded that Anguina funesta tritici and R. iranicus are exclusively associated with Anguina was clearly different from Anguina agrostis and other described tritici; R. toxicus with Anguina funesta and Anguina paludicola species and, thus, the validity of species was confirmed. Anguina Bertozzi & Davies 2009 (Bertozzi and Davies 2009; McKay et al. funesta induces seed galls in the ovaries of some Festuca, Lolium, 1993; Riley and Ophel 1992); R. rathayi with Anguina sp. parasitizing and Vulpia species (Price et al. 1979b; Riley 1995), but only Dactylis glomerata L. (Hardison 1945); and R. festucae with Anguina L. rigidum and V. myuros (L.) C.C. Gmel. are known as hosts from graminis. Cross-inoculation experiments and in vitro adhesion tests natural infestations. ARGT is most common in South Australia and revealed that Anguina australis Steiner 1940, Anguina tritici and an Western Australia, but also occurs in New South Wales. It spread to Anguina sp. from Holcus lanatus L. are potential vectors of some South Africa, most likely in infested seed from Australia, but did not strains of R. toxicus (Riley 1992; Riley and McKay 1990; Riley et al. spread as quickly or have as much economic impact (Subbotin and 2001). Riley 2012). Recently, Anguina funesta was also found in Oregon Little is known about the relationships between the Rathayi- (Meng et al. 2012). bacter sp. and anguinid nematodes. Based on current information, The life cycle of Anguina funesta is typical of other seed gall these relationships should not be considered mutually beneficial nematodes having one generation per year. The second stage since nematodes do not always remain viable in bacterial galls. juveniles (Fig. 5A) become dormant in drying galls, which drop to Infective juvenile nematodes carry bacteria on their cuticle (Fig. the soil surface near as plants senesce and survive anhydrobiotically 3B) in a film of water from galls resting on the soil surface to the until the following season. With rain, juveniles rehydrate, regain growing points of a nearby young plant. The juvenile nematode activity, and invade nearby plants. The juveniles congregate at (Fig. 5A) enters developing ovules, where they induce seed gall the meristem of infested tillers and wait for ovary initiation before formation that the bacteria later colonize. Results of experiments initiating a gall by invading the ovary and modifying its development.

FIGURE 5 Light microscopic photo of A, infective juvenile and B, female of seed gall nematode parasitizing Sporobolus cryptandrus in Idaho. Scale bar 5 50 µm.

810 PHYTOPATHOLOGY Within the gall, second stage juveniles feed and molt through two tunicamycin group of nucleoside antibiotics (Eckardt 1983) that further juvenile stages to become adults (Fig. 5B) (Riley and Barbetti prevent N-linked glycosylation resulting in cell death. The exact 2008). Females and males usually range from a few up to 20 per gall mechanisms of toxin production remain unknown, but related (Riley and Bertozzi 2004). Each female can produce nearly 1,000 eggs systems in the Actinomycetes such as chartreusis (Price et al. 1979a, b). The nematodes develop to second stage juveniles Leach et al. 1953, S. clavuligerus Higgens and Kastner 1971, and inside the eggs and then hatch in the gall. The freshly hatched juveniles Actinosynnema mirums Hasegawa et al. 1978 synthesize tunica- cannot survive desiccation, but mature physiologically to become the mycin in a multistep biosynthetic pathway involving uridine, UDP- survival state as the host senesces and desiccates (Riley and Barbetti GlcNAc, and at least five enzymes (Price and Tsvetanova 2007; 2008). Wyszynski et al. 2010). Anguina paludicola, another seed gall nematode vectoring Previous reports implicate corynetoxin production in R. toxicus R. toxicus, was described by Bertozzi and Davies (2009) from with the presence of a bacteriophage (Ophel et al. 1993). Stynes two hosts: its type host, the nonnative grass Polypogon monspeliensis, and Bird (1983) originally identified a bacteriophage commonly and a native Australian grass, Lachnagrostis filiformis (Forst.) Trinius associated with R. toxicus by using electron microscopy. Initial from areas subject to occasional seasonal flooding in South Australia reports indicated that adding the bacteriophage (NCPPB 3778) to and New South Wales. The life cycle and relationship of Anguina cultures of R. toxicus consistently resulted in the production of paludicola is similar to that of Anguina funesta butwithsome tunicamycin (Ophel et al. 1993). However, later work identified differences that represent adaptations for survival in ecosystems toxin-producing cultures of R. toxicus devoid of the bacteriophage subject to seasonal inundation (Bertozzi and McKay 1995; Davis (Kowalski et al. 2007). The ultimate role of bacteriophage infection et al. 1995; McKay et al. 1993; Riley and Barbetti 2008). in R. toxicus toxin production in nature remains to be determined, although it should be noted that a counterpart phage has not been SURVIVAL POTENTIAL identified for R. iranicus or Rathayibacter sp. EV system. It is unclear whether toxin production is a product of the phage, the bacteria, or an interaction/combination of the two. A complete The production of gummosis or bacterial slime is associated with genome sequence of the phage is available, but no genes related symptoms resulting from infection by Rathayibacter. This gummosis to toxin production were identified as part of the phage DNA is EPS, composed of homopolysaccharides and heteropolysaccharides (W. Schneider, A. Sechler, and E. Rogers, unpublished data). secreted by the bacteria (Donot et al. 2012; Leigh and Coplin 1992). It The evolutionary drivers of developing and maintaining toxin is associated with the cell wall, forming a capsule, or it may become production in R. toxicus are also unclear. Tunicamycin serves as a detached, forming a slime layer (Coplin and Cook 1990; Denny 1995; very effective nematicide, but most members of the genus are able to Fett 1993). R. “agropyri” was isolated in 1982 from samples stored at overcome nematodes and usurp galls without toxin production. In the Washington State University Mycological Herbarium, Pullman, addition, there is a fitness cost to toxin production, as R. toxicus WA that were collected in the 1940s and 1950s (Murray 1986). This is cultures grow slower while producing tunicamycin (Ophel et al. the longest known survival of bacteria on a plant sample and it is 1993). The toxin may serve multiple purposes, acting as a weapon hypothesized that survival was facilitated by the encapsulation of against competing bacteria and other microorganisms, while also R. “agropyri” in the dried EPS, which enables the bacteria to withstand eliminating nematodes from galls. However, this strategy appears to fluctuating environmental conditions (Boles et al. 2004; Franklin et al. be limited to R. toxicus and possibly R. iranicus and Rathayibacter 2011). sp. EV. The triggering of toxin production by the bacteriophage is The seed gall nematode vectors of Rathayibacter spp. feed and also interesting. In this case the bacteriophage may have evolved a reproduce within the developing ovaries of plant seeds and overwinter mechanism for triggering toxin production to increase the success in seed galls as anhydrobiotic juveniles or adults. This dormant stage of its primary obligate host. can survive within the seed gall for many years. For example, Anguina tritici did not lose its ability to invade wheat seedlings after 32 years of dormancy (Limber 1973). In this dehydrated state, nematodes are more DETECTION AND DIAGNOSTICS FOR BACTERIA, tolerant to extreme environmental conditions than are their hydrated NEMATODE, AND TOXIN counterparts. The second stage juveniles of Anguina agrostis in seed galls survived exposure up to 155°C for 5 min, higher than that Methods for specifically detecting R. toxicus in infested plants recorded for any other metazoan (Eisenback et al. 2013). have been published (see below), and several full genomic sequences are available in open access databases. However, the TOXIN PRODUCTION Select Agent status and associated scarcity of biological samples has hampered the validation of a diagnostic assay for official The production of toxin in forage grasses is the result of a confirmation of the presence of the pathogen in the United States. complex interaction between Rathayibacter species, a seed-gall Sampling. As with most field-oriented plant pathogen diagnostic nematode, a susceptible host, and potentially a bacteriophage. To methods, the primary challenges are in sampling and sample date one species, R. toxicus, has been definitively shown to produce preparation. R. toxicus infests forage grasses that may be spread toxin, and two other species, R. iranicus and Rathayibacter sp. EV, across several acres; samples may consist of hay or seed from areas are suspected toxin producers. The infection cycle begins when the where symptomatic animals are identified. The most common bacterium adheres to the cuticle of compatible juvenile nematodes it methods used in Australia involve soaking seed, infested grass or encounters in the soil, and is carried to the growing point of the hay samples in water or buffer and recovering live bacteria for forage grass by the nematode (Riley and McKay 1990). The subsequent analyses. The degree of infestation influences the nematode initiates gall production as a natural part of the life cycle sensitivity of the diagnostic assay. Larger samples result in less and R. toxicus competes with the nematode for the gall. The trigger certainty with regard to diagnosis of the causal agent. for toxin production is unknown, but toxin generally appears late in Morphological and biochemical traits. Like most bacteria, the growing season as seeds are senescing. Senesced seeds dry and Rathayibacter spp. can be identified to the genus level by a fall to the ground with galls colonized by bacteria to repeat the combination of morphological characters, cell wall components disease cycle. and carbon substrate utilization (Burkholder 1948; Evtushenko and The toxins produced by R. toxicus, referred to as corynetoxins, Takeuchi 2006; Zgurskaya et al. 1993). Further distinction may be are a mixture of chemically related glycolipids belonging to the achieved for some species by including specific traits such as salt

Vol. 107, No. 7, 2017 811 intolerance (Riley and Ophel 1992), bacteriocin production (Gross R. rathayi, R. michiganense subsp. insidiosum (sic: Clavibacter and Vidaver 1979) and vancomycin resistance (inferred by presence michiganensis subsp. insidiosus), and Corynebacterium agropyri of vanA gene) (Postnikova et al. 2017) (A. Sechler, unpublished (R. “agropyri”). The improved immunoassay for R. toxicus in hay data). Rathayibacter spp. are not difficult to isolate, but grow slowly, (Masters et al. 2011) is used by Diagnostics and Laboratory and in general more rapid methods are required for diagnosis in order Services, Biosecurity and Regulation Department of Agriculture to respond quickly to a disease outbreak. and Food, Western Australia (DAFWA) and the laboratories at the Nucleic acid-based assays: Bacteria. Although time-consuming, South Australian Research and Development Institute (SARDI) amplified fragment length polymorphism (Agarkova et al. 2006) and (A. Masters, personal communication). PCR-16S rRNA-RFLP (Lee et al. 1997) analyses have been used to Immunoassays: Corynetoxins. Although the corynetoxin characterize the phylogeny of Clavibacter and Rathayibacter,and (tunicamycin antibiotic) responsible for ARGT can be identified could be used to identify Rathayibacter spp. More rapid assays using and detected through chemical analyses such as HPLC and mass methods such as PCR are preferred to accommodate smaller sample spectrometry, these techniques are expensive, time-consuming, size and the ability to rapidly and accurately screen large numbers of and require large and impractical amounts of sample material. A samples. Rathayibacter spp. can be identified by PCR of 16S rRNA standardized protocol using ELISA was developed with monoclo- genes from samples containing live bacteria, followed by sequencing nal antibodies raised against conjugated tunicamycin for more rapid and searches against gene databases. A PCR assay based on the and sensitive toxin identification in hay and veterinary samples R. toxicus 16S rRNA region V9 and bacteriophage NCPPB 3778 (Than et al. 2002). However, this assay is not currently in use by any sequence analysis was developed for detection of R. toxicus and the government. associated phage (Kowalski et al. 2007). However, only one other species in the genus, R. tritici, was used to test whether the target DISEASE MANAGEMENT sequence was unique to R. toxicus. PCR/mass spectrometry was applied to detect and identify multiple species of plant pathogenic It is theoretically possible to control ARGT at any level of the bacteria, including R. iranicus, R. rathayi, R. tritici,andR. toxicus interaction: grazing animal, grass species, nematode, or bacterium (Postnikova et al. 2008). More recently, real-time PCR primers and (Fisher 1977). It is generally not practical to apply nematicides or probes were described to identify the same species from grasses and antibiotics tolarge grazing areas tocontrol the nematode or bacterium grains (Postnikova et al. 2017). directly. In an attempt to immunize animals against tunicamycin The analysis of vancomycin resistant protein vanA, CRISPR- toxicity, researchers at the CSIRO Division of Animal Health in associated protein cse4, secA ATPase, chromosome partition protein Australia injected sheep with tunicamycin conjugated to a carrier SMC, tRNA dihydrouridine synthase, and cysteine desulfurase genes protein (Than et al. 1998). Although the treatment provided and inter-simple sequence repeats allowed identification of three protection against ARGT after consumption of contaminated seed population groups of R. toxicus (Arif et al. 2016). Sequences of the heads in approximately 90% of vaccinated animals, this vaccine gyrB, recA, rpoB, and ppk genes also could be useful for identification does not appear to have been commercialized. Likewise, an antidote of Rathayibacter species (B. K. Schroeder, W. L. Schneider, D. G. was developed, but never commercialized (Riley et al. 2014); in Luster, A. Sechler, and T. D. Murray, unpublished data) (Starodumova both cases the practicality of administering the treatment and their et al. 2014; Vasilenko et al. 2016). effectiveness against high doses of toxin were questionable. Nucleic acid-based assays: Nematode. Diagnosis of the gall- Furthermore, the need for control measures has declined over the forming nematodes based on morphological and morphometrical past 30 years due to a decline in ARGT outbreaks, largely owing to characters is difficult, time consuming, and requires significant success of past management practices including deployment of the expertise. Powers et al. (2001) and Subbotin et al. (2004) molecularly antagonist Dilophospora alopecuri (Fr.) Fr. (Riley 1994). characterized agriculturally important anguinid species and showed Currently used control measures focus on preventing ryegrass the ITS rRNA gene could serve as a reliable marker for identification seed maturation by applying herbicide, mowing and/or heavy of this nematode group. Powers et al. (2001) also showed that PCR- grazing immediately after head emergence, prior to seed set and ITS1-RFLP using AluI, BsrI, EcoRI, HaeIII, HhaI, HinfI, and TaqI maturation when tunicamycin toxicity increases rapidly (https:// could clearlydistinguishAnguina agrostis, Anguina funesta, Anguina www.agric.wa.gov.au/livestock-biosecurity/controlling-annual- pacificae Cid del Prado Vera & Maggenti 1984, Anguina tritici, ryegrass-toxicity-argt-through-management-ryegrass-pasture). This A. agropyronifloris Norton 1965, Anguina microlanae (Fawcett approach must be followed for 2 to 3 years and combined with 1938) Steiner 1940, Afrina wevelli van den Berg 1985, and several planting clean ryegrass seed to reduce the number of nematode and unidentified species from one another. Later, PCR-ITS1-RFLP and bacterial galls as well as the incidence of ARGT. sequencing of this gene region was used to confirm the presence of Anguina funesta in Oregon (Meng et al. 2012). Ma et al. (2011) LOOKING AHEAD developed TaqMan real-time PCR methods for detection of second stage juveniles of Anguina agrostis. Using the ITS rRNA gene Although R. toxicus is a Select Agent in the United States, the fragment, Li et al. (2015) developed and validated TaqMan real-time other five described Rathayibacter species remain relatively PCR methods for detection of Anguina agrostis, Anguina funesta, unstudied because the diseases they cause are obscure and do not Anguina tritici,andAnguina pacificae. Although all these protocols have a recognized economic impact. This situation has resulted in enable fast and accurate identification of these species, comparative knowledge gaps regarding the basic biology of these plant pathogens. sequence analysis of the ITS region should be considered as the main R. toxicus poses a threat to U.S. agriculture because of its ability to approach if test results are inconclusive. produce corynetoxins that are lethal to grazing animals. Two other Immunoassays: Bacteria. An ELISA for detection of R. toxicus species, R. iranicus and the undescribed Rathayibacter sp. EV are was developed against a surface antigen from the bacterium with suspected of being able to produce corynetoxins. The tunicamycin subsequent improvements to sampling and streamlining the assay biosynthetic pathway responsible for corynetoxin production is well (Masters et al. 2006; 2011). A semiquantitative ELISA protocol was characterized from other bacteria, but its regulation and evolutionary later developed for improved risk evaluation (Masters et al. 2014). role in the life cycle of R. toxicus is not understood. A bacteriophage The ELISA protocols and improvements have been applied to was initially implicated in toxin production by R. toxicus, but current identify pastures at high-risk for ARGT, and to screen exports in evidence suggests that the association is not absolute, and there is no Australia since 1996. The monoclonal antibodies raised against the evidence for phage involvement in toxin production by Rathayibacter R. toxicus antigen were screened for specificity against R. tritici, sp. EVor R. iranicus.

812 PHYTOPATHOLOGY Little is known about the potential for toxin production by the Bertozzi, T., and Davies, K. A. 2009. Anguina paludicola sp. n. (Tylenchida: other described Rathayibacter species. Reports of unexplained Anguinidae): The nematode associated with Rathayibacter toxicus infection livestock poisonings in the United States leave open the possibility in Polypogon monspeliensis and Lachnagrostis filiformis in Australia. Zootaxa 2060:33-46. that R. toxicus or a related toxigenic Rathayibacter species may be Bertozzi, T., and McKay, A. C. 1995. Incidence on Polypogon monspeliensis responsible (Riley et al. 2003). These poisonings have occurred of Clavibacter toxicus and Anguina sp., the organisms associated with mostly in the western United States over a long period of time and do ‘flood plain staggers’ in South Australia. Aust. J. Exp. Res. 35:567-569. not have a clear pattern, which makes them very difficult to study. Bird, A. F., and Riddle, D. L. 1984. Effect of attachment of Corynebacterium Preliminary data suggest additional species of both Rathayibacter rathayi on movement of Anguina agrostis larvae. Int. J. Parasitol. 14:503-511. and Anguina exist in the United States (B. L. Barrantes-Infante, Bird, A. F., and Stynes, B. A. 1977. The morphology of a Corynebacterium sp. unpublished data) (B. K. Schroeder, W. L. Schneider, D. G. Luster, parasitic on annual rye grass. Phytopathology 67:828-830. Bird, A. F., Stynes, B. A., and Thomson, W. W. 1980. A comparison of A. Sechler, and T. D. Murray, unpublished data). Consequently, nematode and bacteria-colonized galls induced by Anguina agrostis in important data needed to conduct a risk analysis for establishment Lolium rigidum. Phytopathology 70:1104-1109. of R. toxicus or other toxigenic Rathayibacter species in the United Boles, B. R., Thoendel, M., and Singh, P. K. 2004. Self-generated diversity States are missing and necessitates a need to develop a better produces “insurance effects” in biofilm communities. Proc. Natl. Acad. Sci. understanding of the species of Rathayibacter and their vectors that USA 101:16630-5. are present in the United States. Development and validation of Bryden, W. L., Irwin, C. E., Davis, E. O., Curran, G. L., Lean, I. J., McKay, detection assays for R. toxicus and its vectors that can discriminate A. C., Edgar, J. A., and Burgess, L. W. 1991. Flood plain staggers: An intoxication in cattle due to the ingestion of blown grass (Agrostis avena- among the known and unknown Rathayibacter and their vectors cea). Proc. Nutr. Soc. Aust. 16:240. also are needed to protect U.S. agriculture from its introduction. Burkholder, W. H. 1948. Genus 1. Corynebacterium Lehmann and Neumann. As new Rathayibacter and Anguina species are identified and Pages 381-408 in: Bergey’s Manual of Determinative Bacteriology, 6th ed. characterized, existing detection assays need to be reevaluated to R. S. Breed, E. G. D. Murray, and A. P. Hitchens, eds. The Williams & insure they remain specific for the target pathogen. It also will be Wilkins Co., Baltimore, MD. necessary to determine if these new species are capable of Carlson, R. R., and Vidaver, A. K. 1982. of Corynebacterium plant pathogens, including a new pathogen of wheat, based on polyacrylamide gel producing corynetoxins now or if given the proper circumstances electrophoresis of cellular proteins. Int. J. Syst. Bacteriol. 32:315-326. such as introduction of the corynetoxin biosynthetic gene cluster, Carne, W. M. 1926. Earcockle (Tylenchus tritici) and a bacterial disease could they produce corynetoxins? These are important questions (Pseudomonas tritici) of wheat. J. Dept. Agric. W. Aust. 3:508-512. given that the known Rathayibacter species produce similar disease Carslake, T. 2006. Livestock: Ryegrass toxicity. Farming Ahead 178:62-63. symptoms in several grass species and are found in similar envi- Chizhov, V. N., and Subbotin, S. A. 1990. [Plant-parasitic nematodes of the ronments as R. toxicus. subfamily Anguininae (Nematoda, Tylenchida). Morphology, trophic spe- cialization, system.] Zool. Zh. 69:15-26 (in Russian). Cockrum, P., and Edgar, J. 1985. Rapid estimation of corynetoxins in bacterial ACKNOWLEDGMENTS galls from annual ryegrass (Lolium rigidum Gaudin) by high-performance liquid chromatography. Aust. J. Agric. Res. 36:35-41. Financial support of grants funded by the 2008 Farm Bill, Section Collins,M.D.,andBradbury,J.F.1986. Plant pathogenic species of 10201 administered through the U.S. Department of Agriculture Corynebacterium. Pages 1276-1284 in: Bergey’s Manual of Systematic (USDA) Animal and Plant Health Inspection Service (13-8130- Bacteriology. P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt, 0247-CA and 14-8130-0367-CA) and the Emerging Research eds. Vol. 2. The Williams & Wilkins Co., Baltimore, MD. Coplin, D. L., and Cook, D. 1990. Molecular genetics of extracellular poly- Issues Program funded by the Washington State University saccharide biosynthesis in vascular phytopathogenic bacteria. Mol. Plant- Agricultural Research Center are gratefully acknowledged. PPNS Microbe Interact. 3:271-279. 0739, Department of Plant Pathology, College of Agricultural, Cunningham,I.J.,andHartley,W.J.1959.Ryegrassstaggers.N.Z.Vet.J.7:1-7. Human, and Natural Resource Sciences, Agricultural Research Davis, E. O., Curran, G. E., Hetherington, W. T., Norms, D. N., Wise, G. A., Center, Hatch Project No. WNP00670, Washington State Univer- Rothi, I. J., Seawright, A. A., and Bryden, W. L. 1995. Clinical, patho- sity, Pullman, WA 99164-6430. 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Vol. 107, No. 7, 2017 813 Evtushenko, L. I., Dorofeeva, L. V., Cole, J. R., Subbotin, S. A., and Tiedje, Leigh, J. A., and Coplin, D. L. 1992. Exopolysaccharides in plant-bacterial J. M. 2000. Leifsonia poae gen. nov., sp. nov., isolated from nematode galls interactions. Annu. Rev. Microbiol. 46:307-346. on Poa annua, and reclassification of ‘Corynebacterium aquaticum’ Leif- Li, W., Zonghe, Y., Nakhla, M. K., and Skantar, A. M. 2015. Real-time PCR son 1962 as Leifsonia aquatica (ex Leifson 1962) gen. nov., nom. rev., methods for detection and identification of the nematodes Anguina funesta, comb. nov. and Clavibacter xyli Davis et al. 1984 with two subspecies as A. agrostis, A. tritici, and A. pacificae. Plant Dis. 99:1584-1589. Leifsonia xyli (Davis et al. 1984) gen. nov., comb. nov. Int. J. Syst. Evol. Limber, D. P. 1973. 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